There is a critical need for assays of protein activity in disease models and in patient samples. This need has been created by the advent of molecularly targeted therapies aimed at aberrant signaling pathways now known to underlie cancer and other diseases. The ability to measure enzymatic activity of dysregulated proteins would be extremely advantageous for directing treatment options as well as assessing treatment efficacy in individual patients. A collaborative interdisciplinary research program is proposed to create the instrumentation and chemical tools needed to directly assess the catalytic activity of protein kinases in living cells from disease models and cancer patients. The investigators will further develop and optimize a validated microelectrophoresis platform for the biochemical analysis of ultra small (single cell) samples that utilizes fluorescently labeled substrates for assaying intracellular enzyme activity. Combinatorial libraries will be generated and screened to develop specific kinase substrates. These substrates will be modified to create membrane permeant and long-lived compounds capable of accurately reporting the enzymatic activity of specific kinases within a cytoplasmic milieu. These reporters will be assayed using the microelectrophoresis platform in tumor cell lines to validate the reporters within living cells. Single cells taken directly from patients undergoing surgical resection of pancreatic adenocarcinoma will then be profiled for the enzymatic activity of the patient cells using the technology. Four kinases have been chosen for these feasibility studies: EGFR, ErbB-2, Akt, and S6 kinase. These kinases were chosen by virtue of their pertinence to both research and clinical practice in oncology. Breast and pancreatic cancer have been selected as model diseases for this study by virtue of the state of knowledge in signal transduction for these cancers and their overall importance to public health. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to develop an assay for active signal transduction pathways in the tumor cells of patients. This would enhance diagnostics and prognostics as well aid therapeutic planning. The final aim of the work focuses on pancreatic cancer, a disease with a very poor survival rate.